Cryptococcus neoformans is a fungal pathogen that causes pulmonary and central nervous system infections in immunocompromised and, less commonly, immunocompetent patients. Cryptococcal meningitis (CM) is the most common disease caused by C. neoformans and is a major opportunistic infection affecting people living with HIV/AIDS. Although CM kills people with HIV/AIDS in both developed and resource-limited regions, it causes disproportionately high mortality in resource-limited areas with high HIV prevalence. One reason for this imbalance is that therapies that directly kill C. neoformans are available and practical in resource-advantaged regions and are not in resource-limited regions. One expedient approach to identifying new drugs for a particular disease is to screen molecules that are already approved for use in humans for the desired side activity. Previous studies have shown that a variety of drugs possess antifungal activity despite being used for other indications in the clinic. The goal of this project is to identify previously marketed drugs that have anti-cryptococcal activity using a novel high throughput screening assay specific for fungicidal drugs (Aim 1). As part of our preliminary studies, we have already identified a drug with high activity against Cryptococcus: the estrogen receptor antagonist, toremifene. In order to develop the potential for the promising drugs identified in the screen, we will characterize the in vitro activity of toremifene and other drugs with respect to direct antifungal activity; to their activity in combination with other anti-cryptococcal drugs; and their ability to modify/modulate the virulence properties of Cryptococcus (Aim 2). We will also use a chemical genetic approach to characterizing the molecules mechanism of action (Aim 3). Finally, the in vivo efficacy of toremifene (alone and in combination with fluconazole) will be evaluated using a mouse model of cryptococcosis (Aim 4). PUBLIC HEALTH RELEVANCE: Cryptococcus neoformans is a fungal pathogen that causes pulmonary and central nervous system infections in immunocompromised and, less commonly, immunocompetent patients. Cryptococcal meningitis (CM) is the most common disease caused by C. neoformans and is a major opportunistic infection affecting people living with HIV/AIDS. Although CM kills people with HIV/AIDS in both developed and resource-limited regions, it causes disproportionately high mortality in resource-limited areas with high HIV prevalence. A reason for this imbalance is that therapies that directly kill C. neoformans are available and practical in resource-advantaged regions but are not in resource-limited regions. Since CM is one of the most common AIDS-defining conditions, many patients in resource limited regions must first survive CM in order to take advantage of the increasing availability of highly active anti-retroviral therapy (HAART) for HIV. This project is designed to identify anti-cryptococcal molecules within the set of drugs previously marketed to treat other diseases as a way to rapidly bring an active molecule to clinical use. We will use a novel screen that specifically detects fungicidal anti-cryptococcal activity to screen a collection of ~2000 drugs. The activity of drugs that are positive in the screen will be tested alone and in combination with other anti-cryptococcal drugs and we will study the mechanism of action of highly active molecules. Finally, we have already found that toremifene, an estrogen receptor antagonist used to treat breast cancer, is effective against C. neoformans in the test tube. We will test its activity in a mouse model of cryptococcal infection. If toremifene is active in this model, it could be rapidly moved to clinical trials as a fungicidal drug that is taken by mouth and, thus, useful in resource-limited regions.